Multicomponent reactions, applications in medicinal chemistry & new modalities in drug
discovery
Konstantinidou, Markella
DOI:10.33612/diss.111908148
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Publication date: 2020
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Konstantinidou, M. (2020). Multicomponent reactions, applications in medicinal chemistry & new modalities in drug discovery. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.111908148
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CHAPTER
11
DESIGN AND SYNTHESIS OF PROTEOLYSIS TARGETING
CHIMERAS FOR THE LEUCINE-RICH REPEAT KINASE 2
(LRRK2)
Manuscript in preparation
Markella Konstantinidou, Asmaa Oun,Pragya Pathak, Zefeng Wang, Bidong Zhang, Frans ter Brake, Amalia Dolga, Arjan Kortholt and Alexander Dömling
ABSTRACT
Here we present the rational design and the synthetic methodologies towards proteolysis targeting chimeras (PROTACs) for the recently-emerged Parkinson’s target leucine-rich repeat kinase 2 (LRRK2). Two highly potent, selective and brain-penetrating kinase inhibitors were selected and their structure was appropriately modified to assemble a CRBN PROTAC. Biological data show strong kinase inhibition and the ability to enter the cells for the synthesized compounds. However, data regarding the degradation of the target protein are inconclusive. The reasons for the inefficient degradation of the target are discussed.
INTRODUCTION
Parkinson’s disease (PD) is one of the most common neurodegenerative diseases and although most PD cases are idiopathic and the etiology is largely unknown, environmental and genetic factors are also implicated. Among the implicated genes is the leucine-rich repeat kinase 2 encoding gene encoded by PARK8. LRRK2 mutations have been observed in a number of idiopathic late-onset Parkinson’s patients and are the most common cause of familiar PD.[1-3]
Leucine – rich repeat kinase 2 (LRRK2) is a large protein, consisting of 2527 aminoacids and including multiple domains. Interestingly, it includes both a kinase and a GTPase domain. Regarding the mutations, they mostly occur in the GTPase and the kinase domain, leading to increased kinase activity and autophosphorylation. A significant number of disease-associated LRRK2 mutations have been identified to date among which five missense mutations (R1441C, R1441G, Y1699C, G2019S and I2020T) linked to pathogenesis.[4,5]
Figure 1. Structure of LRRK2.
There have been extensive drug discovery efforts to develop inhibitors for LRRK2, focusing on active site kinase inhibitors in particular. Starting from 2006, three patent reviews have been published, covering the numerous potent scaffolds against the target.[6-8] In 2018, the first clinical
trial (NCT03710707) was announced by Denali Therapeutics Inc., followed by a second clinical trial in 2019 (NCT04056689).[9] However, the structures of DNL201 and DNL151 are not disclosed.
Figure 2. Data deriving from PubMed search with the keywords “LRRK2 inhibitors”. X-axis: year of
publication, Y-axis: number of publication.
RESULTS AND DISCUSSION
Our aim in this project was to evaluate the possibility of degrading LRRK2 using PROTACs, instead of inhibiting the target’s enzyme activity. The starting points for designing PROTACs were known kinase inhibitors. Due to the abundance of scaffolds in the literature the following requirements were considered significant in the choice of the inhibitors:
1. High potency, preferably low nanomolar inhibitors; 2. High selectivity in the kinome;
3. Penetration of the blood-brain barrier;
4. Availability of structural data regarding the binding mode;
5. Solvent exposed functional group to attach the linker without compromising the kinase binding;
6. Number of synthetic steps;
7. Cost and availability of starting materials.
Based on those requirements, two scaffolds were selected: PF-06447475, developed by Pfizer and GNE-7915, developed by Genentech. In particular PF-06447475, has an IC50 of 3 nM in the enzyme assay, 25 nM in the whole cell assay, is brain penetrant, high selective in the kinome and does not show toxicity in rat models. The co-crystal structure with MST3 kinase is reported (PDB 4U8Z).[10] GNE-7915 is also highly potent (IC
50 of 1.9 nM in the enzyme assay, 29 nM in the whole
cell assay), brain penetrant and with high kinome selectivity (1 out of 187).[11] In this case only a
Figure 3. Up: structures of PF-06447475 and GNE-7915. Bottom left: crystal structure of PF-06447475
(magenta sticks) with MST3 (blue cartoon) [PDB 4U8Z], bottom right overlap of docking pose of GNE-7915
(pink sticks) over PF-06447475 (magenta sticks) in MST3 active site (blue cartoon).
The available structural data for PF-06447475 show that the oxygen of the morpholine participates in a hydrogen bond that is important for selectivity among the kinome, however, position 3 of the morpholine is solvent exposed and it could be used to attach a linker. On the contrary for GNE-7915, the morpholine moiety seems to be completely solvent exposed and the hypothesis is that is not a strict requirement for binding.
In order to choose which E3 ligase to target, the expression levels in different tissues were checked in the database of protein atlas.[12] A comparative analysis revealed that cereblon and MDM2 are
expressed in the same parts of the brain, whereas von Hippel Lindau is not. MDM2 is expressed in all tissue in high levels, so for selectivity issues cereblon seemed a better choice to begin with.
Figure 4. Expression levels in different organs of LRRK2, cereblon, VHL and MDM2. The regions of interest
in the brain are within the blue circle. MDM2
VHL CRBN LRRK2
a. Synthetic route for the kinase inhibitors and main intermediates.
The original routes for PF-06447475 and GNE-7915 was followed, however some modifications were considerably improving yields in order to synthesize PROTACs (Scheme 1).
For PF-06447475, the main intermediate (3), was synthesized in 3 steps as shown scheme 1. The original route [10, 13] (upper route) led to intermediate (3) with only 8% overall yield and required
two column purifications. However, by simply changing the protecting group in the first step from (2-chloromethoxyethyl)trimethylsilane (SEM-Cl) to trityl-chloride, as described in a patent[14], the
yield became almost quantitative. Optimization of the Suzuki coupling also increased the yield significantly, and now the optimized route (middle route), led to intermediate (3) with 54% yield over three steps, requiring only one column purification.
Scheme 1. Synthetic routes to reach main intermediate 3 for PF-06447475-based PROTACs, upper (original),
middle (modified) and transformations to intermediates with functional groups (below).
After obtaining intermediate (3), the original inhibitor PF-06447475 (6) is synthesized with a nucleophilic aromatic substitution with morpholine. In order to attach suitable linkers for the PROTACs, morpholines substituted on position 3 were used, bearing either an ester group or a Boc-protected amine to obtain intermediates (7) and (9) respectively. Boc-piperazine was also used in a similar way for intermediate (10). The nucleophilic aromatic substitution was performed under microwave irradiation instead of reflux.
For GNE-7915 the original route [11] includes an amide coupling with morpholine and a nucleophilic
aromatic substitution, with a starting material that also needs to be synthesized. In GNE-7915 the morpholine part is solvent exposed and is also the position where the linkers will be attached. For the benefit of the overall route it is best to introduce the morpholine at the end of the synthesis. We developed an alternative route, starting from the commercially available 4-amino-2-fluoro-5-methoxybenzoic acid (Scheme 2). The initial substitution of the fluorine with a methoxy group, was followed by an esterification and a reduction of the nitro group to receive intermediate (14). The next step was a nucleophilic aromatic substitution using the bifunctional building block 2,4-dichloro-5-(trifluoromethyl)pyrimidine. Interestingly, the substitution can be performed selectively by using zinc chloride as catalyst.[15] The obtained intermediate (15), undergoes a
substitution on the second chloride (16), followed by a hydrolysis to give the carboxylic acid
(17), which is the key intermediate in this synthesis. Overall, the yield is 32% over 6 steps with
only one column purification. The carboxylic acid (17) was used in amide coupling reactions with morpholine to obtain GNE-7915 (11) and also in amide couplings directly with CRBN building blocks or with a substituted morpholine to obtain the amine intermediate (18).
Scheme 2. Synthetic routes to 7915: upper (original), middle (modified) and intermediates for
b. Synthetic routes for cereblon building blocks.
For the synthesis of cereblon building blocks two starting materials were used: the 4-nitroisobenzofuran-1,3-dione and the 4-fluoroisobenzofuran-1,3-dione (Scheme 3).
Scheme 3. Synthetic routes for CRBN-building blocks.
The first synthetic step is the condensation of 4-nitroisobenzofuran-1,3-dione with 3-aminopiperidine-2,6-dione to obtain the nitro-substituted imide (19), which is then reduced to the aniline group to obtain pomalidomide (20). Pomalidomide (20) was then used in an anhydride opening reaction to obtain the carboxylic acid (21). Pomalidomide (20) was also used in an acylation reaction with 2-chloroacetyl chloride to obtain the intermediate (22), which with a substitution reaction led to intermediate (23). In a similar way, the condensation of 4-fluoroisobenzofuran-1,3-dione with 3-aminopiperidine-2,6-dione was performed to obtain the fluoro-substituted imide (24), which underwent a nucleophilic aromatic substitution with linear Boc-protected diamines to obtain intermediates (25) and (26).
c. PROTACs synthesis.
After synthesizing the appropriate kinase intermediates and the cereblon building blocks, the final PROTAC compounds were synthesized with amide coupling reactions. In all the cases of Boc-protected intermediates, the deprotection was performed with HCl in dioxane and the obtained salts were used directly in the amide coupling without purification. An overview of structures and coupling yields are shown in scheme 4.
Scheme 4. Structure of PF-06447475-based-PROTACs and GNE-7915-based PROTACs.
CONCLUSION – FUTURE PERSPECTIVE
In this project, four PF-06447475-based PROTACs and three GNE-7915-based PROTACs, as well as the original inhibitors, as reference compounds, were synthesized. Preliminary biological data show that the compounds are potent kinase inhibitors in in vitro assays. For example compound
able to enter the cells. It is known that incubation of LRRK2 with active site kinase inhibitors results into re-localization of LRRK2 onto microtubules and in the confocal microscopy experiments this is seen as the formation of green filaments. This type of filaments was also observed in the cases of PROTACs that could enter the cells. The hypothesis is that the formation of the filaments probably is affecting the degradation potential, making it more challenging. Moreover, since the full-length structure of LRRK2 is not known, the proximity of lysine residues suitable for ubiquitination and degradation to the kinase site might also not be optimal. Further biological studies are on-going.
REFERENCES
1. W.P. Gilks, P. M. Abou – Sleiman, S. Gandhi, S. Jain, A. Singleton, A.J. Lees, K. Shaw, K.P. Bhatia, V. Bonifiati, N.P. Quinn, J. Lynch, D.G. Healy, J.L. Holton, T. Revesz, N.W. Wood, Lancet 2005, 365(9457), 415 – 416.
2. S. Lesage, S. Janin, E. Lohmann, A.L. Leutenegger, L. Leclere, F. Viallet, P. Pollak, F. Durif, S. Thobois, V. Layet, M. Vidailhet, Y. Agid, A. Dürr, A. Brice, Arch Neurol. 2007, 64(3), 425 – 430.
3. S. Bardien, S. Lesage, A. Brice, J. Carr, Parkinsonism Relat Disord. 2011, 17(7), 501–508. 4. B.I. Giasson, V.M.Van Deerlin, Neurosignals 2008, 16, 99 –105.
5. D.J. Moore, Parkinsonism Relat. Disord. 2008, 14 Suppl 2, S92 – 98.
6. X. Deng, H.G. Choi, S.J. Buhrlage, N.S. Gray, Expert Opin. Ther. Pat. 2012, 22(12), 1415 –1426. 7. R.R. Kethiri, R. Bakthavatchalam, Expert Opin. Ther. Pat. 2014, 24(7), 745 –757.
8. P. Galatsis, Expert Opin. Ther. Pat. 2017, 27(6), 667 – 676. 9. https://clinicaltrials.gov/
10. J.L. Henderson, B.L. Kormos, M.M Hayward, K.J. Coffman, J. Jasti, R.G. Kurumbail, T.T. Wager, P.R. Verhoest, G.S. Noell, Y. Chen, E. Needle, Z. Berger, S.J. Steyn, C. Houle, W.D. Hirst, P. Galatsis, J. Med.
Chem. 2015, 58(1), 419 – 432.
11. A. A. Estrada, X. Liu, C. Baker-Glenn, A. Beresford, D.J. Burdick, M. Chambers, B.K. Chan, H. Chen, X. Ding, A.G. DiPasquale, S.L. Dominguez, J. Dotsonm, J. Drummond, M. Flagella, S. Flynn, R. Fuji, A. Gill, J. Gunzner-Toste, S.F. Harris, T.P. Heffron, T. Kleinheinz, D.W. Lee, C.E. Le Pichon, J.P. Lyssikatos, A.D. Medhurst, J.G. Moffat, S. Mukund, K. Nash, K. Scearce-Levie, Z. Sheng, D.G. Shore, T. Tran, N. Trivedi, S. Wang, S. Zhang, X. Zhang, G. Zhao, H. Zhu, Z.K. Sweeney, J. Med. Chem. 2012, 55(22), 9416 – 9433.
12. https://www.proteinatlas.org/
13. P. Galatsis, (Pfizer), US2017002000A1, 2017. 14. Y-L. Chen, (Novartis), WO2010015637A1, 2010. 15. J.C. Kath, (Pfizer), WO2005023780A1, 2005.
EXPERIMENTAL SECTION
Experimental procedures
Procedure A1 (Protecting step with SEM-Cl): in a 500ml round bottom flash
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (17.9 mmol, 1 equiv) was dissolved in dry THF (120 ml). Cooling in an icebath and addition of NaH (19.69 mmol, 1.1 equiv) in portions as solid. The reaction mixture was stirred at 0oC for 1 h and then (2-chloromethoxyethyl)trimethylsilane (SEM-Cl) (19.3 mmol,
1.08 equiv) was added dropwise. After the addition, the reaction mixture was allowed to reach rt. Stirring at rt for 4h. The reaction was quenched with saturated NaHCO3 (120 ml). THF was removed under reduced pressure and the residue was extracted with EtOAc (100 ml x 3). The combined organic phases were dried over MgSO4, filtered and the crude product was purified by column chromatography (PE – EtOAc, 0 – 20% EtOAc in PE).
Procedure A2 (Protecting step with trityl-Cl): in a round bottom flash
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (17.9 mmol, 1 equiv) was suspended in dry CHCl3 (45 ml). Triethylamine was added (27 mmol, 1.5 equiv) and the suspension was cooled at 0oC. Trityl chloride (21.48 mmol,
1.2 equiv) was added in portions as solid. The reaction mixture was stirred at 0 oC for 15 min under
CaCl2 tube and then at rt for 1h. Solvent was removed under reduced pressure. 100ml MeOH were added in the residue and the formed solid was filtered under reduced pressure and dried under vacuum.
Procedure B1 (Suzuki coupling on intermediate 1): in a 3-neck 500 ml round bottom flask
4-chloro-5-iodo-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (1) (7.1 mmol, 1 equiv), (3-cyanophenyl)boronic acid (7.81 mmol, 1.1 equiv) and K2CO3 (21.3 mmol, 3 equiv) were dissolved in a mixture of DME – H2O (4:1, 100ml). The reaction mixture was degassed for 15min and then Pd(dppf)Cl2 (0.355mmol, 0.05 equiv) was added in one portion. The reaction mixture was heated at reflux for 3h, under N2 flow. Then, it was allowed to reach rt and was diluted with saturated NaCl (100ml) and it was extracted with EtOAc (100 ml x 3). The combined organic phases were dried over MgSO4, filtered and the crude product was purified by column chromatography (PE – EtOAc, 0 – 20% EtOAc in PE).
Procedure B2 (Suzuki coupling on intermediate 4): In a 3-neck round bottom flask
4-chloro-5-iodo-7-trityl-7H-pyrrolo[2,3-d]pyrimidine (4) (9.6 mmol, 1 equiv) and (3-cyanophenyl) boronic acid (19.2 mmol, 2 equiv) were suspended in a 5:1 mixture toluene : EtOH (15 ml : 3 ml), followed by the addition of a saturated solution of NaHCO3 (18 ml). The reaction mixture was degassed for 15 min and then Pd(dppf)Cl2 (0.0384 mmol, 0.004 equiv) was added in one portion. The reaction mixture was heated overnight at 85 oC under N
2 flow. The next day, the reaction mixture was
allowed to reach rt, H20 was added and the reaction mixture was extracted with EtOAc (x3). The combined organic phases were washed with 1N NaOH (x2), Brine (x3), dried over MgS04, filtered
and the solvents were removed under vacuum. The crude product was purified by column chromatography (PE – EtOAc, 0 – 20% EtOAc in PE).
Procedure C1 (Deprotection of SEM-group): 3-(4-chloro-7-((2-(trimethylsilyl)ethoxy)
methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile (2) (2 mmol, 1 equiv) was dissolved in 5ml TFA. Stirring rt overnight under CaCl2 tube. DCM was added (10 ml) and the solvents were removed under reduced pressure to get a yellow oil. 15 ml of methanol were added to get a yellow suspension. Under stirring at 0oC, solid K
2CO3 was added in small portions until pH>12. Methanol was removed
under reduced pressure and water was added (5 ml). The suspension was filtered under vacuum and the obtained solid was washed with water and dried under vacuum.
Procedure C2 (Deprotection of trityl-group): 4-chloro-5-iodo-7-trityl-7H-pyrrolo[2,3-d]
pyrimidine (5) (5.1mmol, 1 equiv) was dissolved in DCM (20 ml). The solution was cooled in an ice-bath and TFA (10 ml) was added. Stirring at 0oC for 10 min and then rt overnight under CaCl
2 tube.
Solvents were removed under reduced pressure. The oily residue was dissolved in 20 ml MeOH. Under stirring at 0oC, solid K
2CO3 was added in small portions until pH>12. Methanol was removed
under reduced pressure and water was added (5 ml). The suspension was filtered under vacuum and the obtained solid was washed with water and dried under vacuum. The obtained solid was triturated with diethylether to remove impurities.
Procedure D1 (Nucleophilic aromatic substitution with morpholine):
3-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile (3) (0.39 mmol, 1 equiv) was suspended in tert-butanol (5 ml). DIPEA (0.78 mmol, 2 equiv) and morpholine (0.43 mmol, 1.1 equiv) were added and the reaction mixture was heated at reflux for 3 h. Solvent was removed and the crude was purified by column chromatography (DCM – MeOH, 0 – 6% MeOH in DCM).
Procedure D2 (Nucleophilic aromatic substitution with 3-substituted morpholines or Boc-piperazine): in a microwave vial 3-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile (3) (0.39
mmol, 1 equiv) was suspended in ethanol (2 ml). DIPEA (0.78 mmol, 2 equiv) and the appropriate secondary amine (0.43 mmol, 1.1 equiv) were added and the reaction mixture was subjected to microwave irradiation (1 h, 150oC). Solvent was removed and the crude was purified by column
chromatography (DCM – MeOH, 0 – 6% MeOH in DCM).
Procedure E (Ester hydrolysis): the ethyl ester (1 equiv) was suspended in a mixture of THF – H2O (2:1, 0.2M) and LiOH (2 equiv) was added. The reaction mixture was stirred rt overnight. Solvents were removed under reduced pressure and the residue was dissolved in 5ml H2O. Cooling at 0oC
and acidification with 2N HCl until pH = 1. Extraction with EtOAc (50 ml x3), drying over MgSO4 ,filtration and evaporation under reduced pressure.
Procedure F (Substitution of fluorine with methoxy group): In a round bottom flask,
2,5-difluoro-4-nitrobenzoic acid (24.6 mmol, 1 equiv) was dissolved in methanol (80 ml) at room temperature. A solution of freshly prepared KOH (73.8 mmol, 3 equiv) in 30 ml MeOH was added dropwise over 20 min. The formed suspension was stirred at room temperature for 2 h and eventually the reaction mixture became a yellow solution. Methanol was removed under reduced pressure and the residue was suspended in 50 ml of EtOAc. While cooling in an ice-bath acidification with 2 N aqueous HCl, until pH =1 and extraction with EtOAc (80 ml x3), drying over MgSO4, filtration and evaporation under reduced pressure.
Procedure G (Esterification): 2-fluoro-5-methoxy-4-nitrobenzoic acid (12) (9.3 mmol, 1 equiv)
was dissolved in ethanol under stirring. N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 11.2 mmol, 1.2 equiv) was added in portions. The reaction mixture was heated at reflux for 5 h. Solvent was removed under reduced pressure and the residue was dissolved in EtOAc (100 ml) and washed with 1N HCl (x2), H2O (x2) and Brine (x2). The organic phase was dried over MgSO4, filtered and the solvent was removed under reduced pressure.
Procedure H (Reduction with stannous chloride): ethyl 2-fluoro-5-methoxy-4-nitrobenzoate (13) (9.1 mmol, 1 equiv) was suspended in a mixture of ethanol – water (65 ml : 6.5 ml). Stannous
chloride (36.4 mmol, 4 equiv) was added in portions at room temperature. The reaction mixture was heated at reflux for 4 h. Solvent was removed under reduced pressure and the residue was diluted with 50 ml EtOAc. Under stirring, saturated NaHC03 was added until pH=8. Extraction with EtOAc (80 ml x3). The combined organic phases were dried over MgSO4, then passed through a pad of celite and the solvent was removed under reduced pressure.
Procedure I (Selective nucleophilic aromatic substitution of chlorine): ethyl
4-amino-2-fluoro-5-methoxybenzoate (14) (9.0 mmol, 1 equiv) was dissolved in a mixture of diethylether (6.0 ml), tert-butanol (4.0 ml) and DCM (15 ml) and was then cooled in an ice-bath. At 0oC under
stirring, zinc chloride (18.0 mmol, 2 equiv) was added in portions as solid, followed by the addition of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (9.0 mmol, 1.0 equiv) and triethylamine (9.9 mmol, 1.1 equiv). The reaction mixture was stirred at 0oC for 1h under CaCl
2 tube and then at room
temperature for 48h. The reaction was monitored by TLC (PE: EtOAc 8:2). The reaction mixture was diluted with 40 ml DCM and slowly 40 ml of water were added (bubbling was observed). Stirring rt for 15 min and then extraction with DCM (50 ml x3). The combined organic phases were washed with Brine, dried over MgSO4, filtered and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography PE: EtOAc (0-50% EtOAc in PE).
Procedure J (Nucleophilic aromatic substitution of chlorine): ethyl
4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxybenzoate (15) (3.3 mmol, 1 equiv) was dissolved in 15 ml dry THF. Cooling at 0oC and then dropwise addition of a 2 N solution of
ethanamine (6.6 mmol, 2.2 equiv) under N2 flow. The reaction mixture was stirred at 0oC for 30 min
and then rt for 2 h. Solvent was removed under reduced pressure. The residue was diluted with EtOAc (50 ml) and washed with H2O (x2) and Brine (x2). The organic phase was dried over MgSO4, filtered and the solvent was removed under reduced pressure.
Procedure K (Synthesis of substituted 2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-diones):
In a round-bottom flask, the appropriate 4-substituted-isobenzofuran-1,3-dione (1 equiv), 3-aminopiperidine-2,6-dione hydrochloride (1 equiv) and sodium acetate (1.2 equiv) were mixed in AcOH (20 ml for 5mmol scale). The resulting mixture was heated at 120 oC overnight. After
cooling to room temperature, most of the AcOH was removed under reduced pressure and the residue was taken in water, filtered and washed with water and dried with vacuum to obtain the crude solid compound.
Procedure L (Reduction): To a solution of
2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (8 mmol, 1.0 equiv) in dry DMF (50 ml) was added the Pd/C (1.6 mmol, 0.2 equiv) under N2. The reaction mixture was hydrogenated with 3.0 atm H2 pressure at room temperature for 4 h. The progress of reaction was monitored by TLC. The reaction mixture was filtered over a pad of celite. The filtrate was diluted with EtOAc and the organic phase was washed with H2O and Brine (x3) and was dried over MgSO4 and filtered. The solvent was removed under reduced pressure, to obtain a solid, which was used directly in the next step.
Procedure M (Anhydride opening): A mixture of 4-amino-2-(2,6-dioxopiperidin-3-yl)
isoindoline-1,3-dione (7.3 mmol, 1.0 equiv), potassium acetate (29.3 mmol, 4.0 equiv) and the appropriate anhydride (29.23 mmol, 4.0 equiv) in glacial AcOH (60 ml) was heated at reflux under nitrogen for 3h. After cooling down, acetic acid was removed under reduced pressure and the residue was extracted with (EtOAc – H2O). The organic phases were dried with MgSO4, filtered and solvents were removed under reduced pressure. The crude product was purified by column chromatography (DCM – MeOH, 0 – 10% MeOH in DCM).
Procedure N (Amidation): To a stirred suspension of 4-amino-2-(2,6-dioxo(3-piperidyl))
isoindoline-1,3-dione (5.00 mmol, 1 equiv) in THF (30 ml), chloroacetyl chloride (5.5 mmol, 1.1 equiv) was added. The mixture was heated to reflux for 30 minutes. The solvent was evaporated under reduced pressure and the obtained solid was treated with diethyl ether (20 ml) and filtered to give the product, which was used directly in the next step.
Procedure O (Aliphatic substitution): A mixture of
2-chloro-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (3.44 mmol, 1.0 equiv ), tert-butyl (piperidin-4-ylmethyl) carbamate (3.8 mmol, 1.1 equiv), NaI (3.44 mmol, 1.0 equiv) and K2CO3 (6.88 mmol, 2 equiv) in THF (30 ml) was stirred at room temperature overnight. The solvent was removed under reduced pressure, water (50 ml) was added, and the reaction mixture was extracted with EtOAc (3 × 100 ml). The combined
under reduced pressure. The crude product was purified by column chromatography (DCM: MeOH = 20:1).
Procedure P (Nucleophilic aromatic substitution of fluorine): The appropriate mono-Boc
protected diamine (1.13 mmol, 1.1 equiv) was added to a stirred solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1.03 mmol, 1.0 equiv) in DMF (1 M) and DIPEA (2.06 mmol, 2.0 equiv). The reaction mixture was stirred at 90 °C for 12 h. Then the mixture was cooled to room temperature, poured into H2O, and extracted twice with EtOAc (3 x 50 ml). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and filtered. Solvents were removed under reduced pressure and the crude compound was purified by column chromatography (PE – EtOAc 0 - 50% EtOAc in PE).
Procedure Q1 (Amide coupling with Boc-protected amines): The appropriate Boc-protected
amine (1 equiv) was deprotected with 4N HCl in dioxane with stirring rt overnight. The reaction mixture was dried under vacuum. Diethylether was added (x2) and was removed under reduced pressure. The obtained HCl salt was used directly in the amide coupling. The HCl salt (1 equiv) was suspended in CHCl3 (0.1M). Under stirring DIPEA (2 equiv) was added, followed by the addition of the carboxylic acid (1 equiv) and EEDQ (2 equiv). The reaction mixture was heated at reflux for 2h. Then it was allowed to reach rt and was purified directly by column chromatography (DCM – MeOH, 0 – 10% MeOH in DCM).
Procedure Q2 (Amide coupling with secondary amines): the carboxylic acid (1 equiv) and
EEDQ (2 equiv) were stirred at rt in CHCl3 (0.1 M), followed by the addition of the secondary amine (1 equiv). The reaction mixture was heated at reflux for 2h. Then it was allowed to reach rt and was purified directly by column chromatography (DCM – MeOH, 0 – 10% MeOH in DCM).
Characterization data
4-Chloro-5-iodo-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (1)
Obtained using procedure A1 on 17.9 mmol scale; 2.9 g, 7.1 mmol, yield 40 %, white solid. 1H NMR (500 MHz, DMSO-d
6) δ 8.69 (s, 1H), 8.14 (s, 1H), 5.60 (s, 2H),
3.51 (t, J = 8.0 Hz, 2H), 0.82 (t, J = 8.0 Hz, 2H), -0.10 (s, 9H). 1H NMR is in good
agreement with published data. [s1]
3-(4-Chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl) benzonitrile (2)
Obtained using procedure B1 on 7.1 mmol scale; 800 mg, 2.1 mmol, yield 30 %, off-white solid. 1H NMR (500 MHz, DMSO-d
6) δ 8.75 (s, 1H), 8.13 (s, 1H), 8.01 (b, 1H),
7.90 – 7.86 (m, 2H), 7.72 – 7.66 (m, 1H), 5.70 (s, 2H), 3.59 (t, J = 8.0 Hz, 2H), 0.86 (t, J = 8.0 Hz, 2H), -0.08 (s, 9H). 1H NMR is in good agreement with published data.[s1]
3-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile (3)
Obtained using procedure C1 on 2.0 mmol scale; 305 mg, 1.2 mmol, yield 60 %, white solid. Obtained also using procedure C2 on 5.1 mmol scale; 1.2 g, 4.6 mmol, yield 90%, white solid. 1H NMR (500 MHz, DMSO-d
6) δ 8.48 (s, 1H), 7.94
(s, 1H), 7.87 – 7.85 (m, 2H), 7.71 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H).1H NMR is
in good agreement with published data.[s1] HRMS (ESI): m/z calcd for C 13H8N4Cl
[M+H]+: 255.0432; found 255.0432.
4-Chloro-5-iodo-7-trityl-7h-pyrrolo[2,3-d]pyrimidine (4)
obtained using procedure A2 on 17.9 mmol scale; 9.3 g, 17.85 mmol, yield 99%, white solid. 1H NMR (500 MHz, CDCl
3) δ 8.27 (s, 1H), 7.38 (s, 1H), 7.31– 7.28 (m, 10H),
7.13 – 7.11 (m, 5H).1H NMR is in good agreement with published data.[s2]HRMS
4-Chloro-5-iodo-7-trityl-7h-pyrrolo[2,3-d]pyrimidine (5)
obtained using procedure B2 on 9.6 mmol scale; 2.8 g, 5.7 mmol, yield 60 %, white solid. 1H NMR (500 MHz, CDCl 3) δ 8.35 (s, 1H), 7.77 – 7.74 (m, 2H), 7.66 – 7.63 (m, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.34 – 7.30 (m, 10H), 7.20 – 7.18 (m, 6H). 13C NMR (126 MHz, CDCl 3) δ 152.7, 152.0, 150.0, 141.6, 134.9, 134.2, 133.5, 131.8, 131.4, 130.8, 130.6, 130.0, 129.8, 129.7, 128.7, 128.0, 127.8, 118.6, 116.1, 113.8, 112.2, 76.8.HRMS (ESI): m/z calcd for C32 H22 N4 Cl [M+H]+ :497.1528; found 497.1525. 3-(4-Morpholino-7h-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile (6) [pf-06447475]
obtained using procedure D1 on 0.39 mmol scale; 32 mg, 0.10 mmol, yield 26 %, white solid. 1H NMR (500 MHz, CDCl
3) δ 10.54 (s, 1H), 8.54 (s, 1H), 7.86 (s, 1H), 7.78
(d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.28 (b, 1H), 3.56 – 3.54 (m, 4H), 3.32 – 3.30 (m, 4H). HRMS (ESI): m/z calcd for C17H16ON5 [M+H]+ :
306.1349; found 306.1344.
Ethyl 4-(5-(3-cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)morpholine-2-carboxylate (7)
obtained using procedure D2 on 0.39 mmol scale; 45 mg, 0.12 mmol, yield 30 %, colourless oil. 1H NMR (500 MHz, CDCl
3) δ 11.79 (s, 1H), 8.56 (s,
1H), 7.85 - 7.84 (m, 1H), 7.79 – 7.77 (m, 1H), 7.63 – 7.62 (m, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.36 (b, 1H), 4.16 (q, J = 7.1 Hz, 2H), 4.04 (dd, J = 9.9, 2.8 Hz, 1H), 3.97 – 3.95 (m, 1H), 3.89 (dd, J = 8.9, 2.7 Hz, 1H), 3.59 – 3.56 (m, 1H), 3.50 (td, J = 11.3, 2.4 Hz, 1H), 3.08 – 3.02 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). HRMS (ESI): m/z calcd for C20H20O3N5 [M+H]+ :378.1561; found 378.1555.
4-(5-(3-Cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)morpholine-2-carboxylic acid (8)
obtained using procedure E on 0.12 mmol scale; 30 mg, 0.08 mmol, yield 65 %, yellow solid.1H NMR (500 MHz, DMSO-d
6) δ 12.37 (s, 1H), 8.42
(s, 1H), 8.01 (b, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.72 - 7.71 (m, 1H), 7.66 (t, J = 7.8 Hz, 1H), 4.02 – 4.00 (m, 1H), 3.73 – 3.71 (m, 2H), 3.04 – 2.99 (m, 2H), 2.87 – 2.84 (m, 2H). HRMS (ESI): m/z calcd for C18H16O3N5 [M+H]+ :350.1248; found 350.1244.
Tert-butyl((4-(5-(3-cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)morpholin-2-yl)methyl)
carbamate (9)
obtained using procedure D2 on 0.39 mmol scale; 52 mg, 0.12 mmol, yield 30 %, yellow solid.1H NMR (500 MHz, CDCl
3) δ 12.24 (s, 1H), 8.52 (s, 1H), 7.82 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.34 (b, 1H), 4.81 (b, 1H), 3.72 – 3.69 (m, 2H), 3.61 – 3.59 (m, 1H), 3.47 – 3.42 (m, 2H), 3.17 – 3.15 (m, 1H), 2.94 – 2.90 (m, 2H), 2.71 – 2.68 (m, 1H), 1.43 (s, 9H).13C NMR (126 MHz, MeOD) δ 161.7, 158.2, 154.2, 151.6, 138.0, 134.0, 132.9, 130.9, 124.4, 119.8, 116.2, 113.7, 104.4, 80.2, 75.6, 67.0, 53.4, 50.4, 43.6, 28.8. HRMS (ESI): m/z calcd for C23H27O3N6 [M+H]+ :435.2139; found 435.2135.
Tert-butyl 4-(5-(3-cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)piperazine-1-carboxylate
(10)
Obtained using procedure D2 on 2 mmol scale; 240 mg, 0.6 mmol, yield 30 %, white solid.1H NMR (500 MHz, CDCl 3) δ 11.04 (s, 1H), 8.52 (s, 1H), 7.84 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.30 (s, 1H), 3.27 (b, 8H), 1.43 (s, 9H).13C NMR (126 MHz, CDCl 3) δ 160.3, 154.5, 153.5, 150.9, 136.5, 132.5, 131.7, 130.1, 129.5, 121.8, 118.7, 115.3, 112.8, 103.2, 80.1, 49.5, 43.3, 28.4. (4-((4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxyphenyl) (morpholino)methanone (11) [GNE-7915]
Obtained using procedure Q2 on 0.26 mmol scale; 46 mg, 0.103 mmol, yield 40 %, white solid. 1H NMR (500 MHz, CDCl
3) δ 8.44 (d, J = 12.3 Hz, 1H),
8.19 (s, 1H), 7.85 (s, 1H), 6.91 (d, J = 5.9 Hz, 1H), 5.22 (b, 1H), 3.91 (s, 3H), 3.80 – 3.77 (m, 4H), 3.68 – 3.66 (m, 2H), 3.62 – 3.59 (m, 2H), 3.45 – 3.43 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H). 1H NMR is in good agreement with published data. [s3]13C NMR (126 MHz, CDCl
3) δ 165.5, 160.3, 158.8, 154.4 (d, J=5.0 Hz), 152.3
(d, J =238.5 Hz), 144.1, 131.8 (d, J =12.3 Hz), 124.7 (q, J =270.3 Hz), 114.4 (d, J =19.2 Hz), 109.7 (d, J =5.0 Hz), 105.6 (d, J =31.8 Hz), 99.7, 66.8, 56.2, 47.7, 42.7, 36.4, 14.4. HRMS (ESI): m/z calcd for C19H22O3N5F4 [M+H]+ :444.1653; found
444.165.
2-Fluoro-5-methoxy-4-nitrobenzoic acid (12)
obtained using procedure F on 24.6 mmol scale; 4.87 g, 22.6 mmol, yield 92 %, light yellow solid. 1H NMR (500 MHz, CDCl
3) δ 7.72 (d, J = 5.5 Hz, 1H), 7.66 (d, J =
9.2 Hz, 1H), 4.02 (s, 3H). 13C NMR (126 MHz, CDCl
3) δ 167.2 (d, J = 3.8 Hz), 155.0 (d,
J = 260.3 Hz), 148.4 (d, J = 3.2 Hz), 142.5 (d, J = 7.3 Hz), 121.6 (d, J = 11.1 Hz), 116.9,
Ethyl 2-fluoro-5-methoxy-4-nitrobenzoate (13)
obtained using procedure G on 9.3 mmol scale; 2.2 g, 9.1 mmol, yield 98 %, off-white solid. 1H NMR (500 MHz, CDCl 3) δ 7.64 – 7.62 (m, 2H), 4.44 (q, J = 7.1 Hz, 2H), 3.99 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl 3) δ 162.9 (d, J = 4.3 Hz), 154.2 (d, J = 257.5 Hz), 148.4 (d, J = 3.0 Hz), 141.5 (d, J = 8.1 Hz), 123.51 (d, J = 12.0 Hz), 116.5, 114.4 (d, J = 29.0 Hz), 62.3, 57.1, 14.1. Ethyl 4-amino-2-fluoro-5-methoxybenzoate (14)
obtained using procedure H on 9.1 mmol scale; 1.92 g, 9.0 mmol, yield 98 %, light orange solid. 1H NMR (500 MHz, CDCl
3) δ 7.27 (d, J = 6.4 Hz, 1H), 6.37 (d, J = 11.9 Hz, 1H), 4.34 (q, J = 7.1 Hz, 4H), 3.86 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 164.90 (d, J = 4.3 Hz), 158.4, (d, J = 252.8 Hz), 142.4 (d, J = 12.1 Hz), 142.25 (d, J = 1.4 Hz), 111.92 (d, J = 2.8 Hz), 106.0 (d, J = 10.8 Hz), 101.4 (d, J = 28.4 Hz), 60.65, 55.95, 14.38. Ethyl 4-amino-2-fluoro-5-methoxybenzoate (15)
obtained using procedure I on 9.0 mmol scale; 1.4 g, 3.6 mmol, yield 40%, off-white solid. 1H NMR (500 MHz, CDCl 3) δ 8.66 (s, 1H), 8.40 (d, J = 12.9 Hz, 1H), 8.29 (s, 1H), 7.40 (d, J = 6.3 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 1.40 (t, J = 7.1 Hz, 2H). 13C NMR (126 MHz, CDCl 3) δ 164.3 (d, J =4.3 Hz), 159.6, 159.4, 157.3 (q, J = 4.9 Hz), 157.0 (d, J = 252.8 Hz), 143.4, 132.5 (d, J = 12.2 Hz), 122.3 (q, J = 272.2 Hz),111.7, 115.1 (d, J = 34.2 Hz), 111.6 (d, J = 1.6 Hz), 107.3 (d, J = 32.1 Hz), 99.8, 61.3, 56.4, 14.3. HRMS (ESI): m/z calcd for C15H13O3N3ClF4 [M+H]+
:394.0576; found 394.0572.
Ethyl 4-amino-2-fluoro-5-methoxybenzoate (16)
obtained using procedure J on 3.3 mmol scale; 1.2 g, 2.9 mmol, yield 88 %, off-white solid. 1H NMR (500 MHz, CDCl 3) δ 8.48 (d, J = 13.7 Hz, 1H), 8.19 (s, 1H), 7.93 (s, 1H), 7.37 (d, J = 6.4 Hz, 1H), 5.24 (s, 1H), 4.38 (q, J = 7.1 Hz, 2H ), 3.94 (s, 3H), 3.63 - 3.59 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.2 Hz, 3H).13C NMR (126 MHz, CDCl 3) δ 164.6 (d, J = 4.3 Hz), 160.2, 158.9, 157.3 (d, J = 251.5 Hz), 154.6 (q, J = 5.3 Hz), 143.1, 134.4 (d, J = 12.9 Hz), 124.7 (q, J = 270.4 Hz), 111.2, 109.7 (d, J = 11.6 Hz), 106.4 (d, J = 32.6 Hz), 99.8, 61.1, 56.3, 36.5, 14.4, 14.3. HRMS (ESI): m/z calcd for C17H19O3N4F4 [M+H]+ :403.1388;
found 403.1385.
4-((4-(Ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxybenzoic acid (17)
Obtained using procedure E on 1.85 mmol scale; 670 mg, 1.8 mmol, yield 97 %, yellow solid. 1H NMR (500 MHz, DMSO-d
6) δ 12.93 (s, 1H), 8.35 (d, J = 13.7 Hz, 1H), 8.26 (s, 1H), 8.16 (s, 1H), 7.46 (t, J = 5.3 Hz, 1H), 7.36 (d, J = 6.7 Hz, 1H), 3.91 (s, 3H), 3.50 – 3.47 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 164.8 (d, J = 3.5 Hz), 160.0, 157.8, 156.2 (d, J = 250.0 Hz), 154.6, 143.5, 133.8 (d, J = 12.5 Hz), 124.6 (q, J = 270.3 Hz), 111.8, 110.3 (d, J = 11.5 Hz), 106.4 (d, J = 23.2 Hz), 99.5 (d, J = 32.0 Hz), 56.4, 35.7, 14.2. HRMS (ESI): m/z calcd for C15H15O3N4F4 [M+H]+ :375.1075; found 375.1071.
Tert-butyl((4-(4-((4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxybenzoyl)morpholin-2-yl)methyl)carbamate (18)
Obtained using procedure Q2 on 0.26 mmol scale; 55 mg, 0.11 mmol, yield 40 %, yellow oil. 1H NMR (500 MHz, CDCl
3) δ 8.42 (t, J = 11.1 Hz, 1H), 8.18 (s, 1H), 7.90 (s, 1H), 6.88 (s, 1H), 5.24 (s, 1H), 4.91 (s, 1H), 4.56 (t, J = 14.4 Hz, 1H), 3.91 (s, 3H), 3.60 – 3.49 (m, 6H), 3.37 – 3.21 (m, 2H), 3.03 – 2.94 (m, 2H), 1.38 (s, 9H), 1.31 (b, 3H).13C NMR (126 MHz, CDCl 3) δ 165.6 (d, J = 12.7 Hz), 160.2, 158.8, 155.8, 154.4 (q, J = 4.9 Hz), 152.2 (d, J = 239.0 Hz), 144.2, 132.0 (d, J = 12.9 Hz), 124.7 (q, J = 269.7 Hz), 114.0, 109.7, 105.6 (d, J = 30.4 Hz), 99.8, 79.5, 74.9, 66.6, 56.2, 47.1, 42.4, 42.1, 36.429.6,
28.3, 28.2, 14.4. HRMS (ESI): m/z calcd for C25H33O5N6F4 [M+H]+: 573.2443; found 573.244. 2-(2,6-Dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (19)
obtained using procedure K on 10 mmol scale; 2.7 g, 8.9 mmol, yield 90 %, purple solid. The crude product was used directly in the next step.1H NMR
(500 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.36 (dd, J = 8.1, 0.7 Hz, 1H), 8.25 (dd, J = 7.5, 0.5 Hz, 1H), 8.14 – 8.11 (m, 1H), 5.21 (dd, J = 12.9, 5.4 Hz, 1H), 2.90 (ddd, J = 17.3, 14.0, 5.4 Hz, 1H), 2.64 – 2.61 (m, 1H), 2.56– 2.52 (m, 1H), 2.11 -2.06 (m, 1H).13C NMR (126 MHz, DMSO-d 6) δ 172.8, 169.6, 165.3, 162.6, 144.5, 136.9, 133.1, 129.0, 127.4, 122.6, 49.5, 30.9, 21.8. 4-Amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (20)
obtained using procedure L on 8 mmol scale; 2.0 g, 7.3 mmol, yield 92 %, yellow solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.09 (s, 1H), 7.46 (dd, J = 8.4,
7.0 Hz, 1H), 7.02 – 6.99 (m, 2H), 6.52 (b, 2H), 5.04 (dd, J = 12.7, 5.4 Hz, 1H), 2.88 (ddd, J = 17.0, 13.9, 5.5 Hz, 1H), 2.54 – 2.50 (m, 1H), 2.04 – 2.00 (m, 1H).13C NMR
5-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-5-oxopentanoic acid (21)
Obtained using procedure M on 7.3mmol scale; 904 mg, 2.4 mmol, yield 32 %, white solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.14 (s, 1H), 9.73 (s, 1H), 8.43 (d, J = 8.3 Hz, 1H), 7.84 – 7.81 (m, 1H), 7.61 (d, J = 7.3 Hz, 1H), 5.14 (dd, J = 12.9, 5.4 Hz, 1H), 2.92 – 2.85 (m, 1H), 2.62 – 2.50 (m, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.08 – 2.05 (m, 1H), 1.86 – 1.80 (m, 2H).13C NMR (126 MHz, DMSO-d 6) δ 174.0, 172.7, 171.5, 169.7, 167.5, 166.6, 136.3, 136.0, 131.4, 126.5, 118.3, 117.2, 48.8, 35.5, 32.7, 30.7, 21.9, 20.1. HRMS (ESI): m/z calcd for C18H18O7N3 [M+H]+:388.1139; found 388.114.
2-Chloro-n-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (22)
obtained using procedure N on 5 mmol scale; 1.61 g, 4.6 mmol, yield 92 %, yellow solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.03 (s, 1H), 10.20 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.57 – 7.51 (m, 2H), 5.16 (dd, J = 13.3, 5.1 Hz, 1H), 4.32 (s, 2H), 2.95 – 2.88 (m, 1H), 2.62 – 2.59 (m, 1H), 2.34 (dd, J = 13.1, 4.4 Hz, 1H), 2.04 – 2.00 (m, 1H).13C NMR (126 MHz, DMSO-d6) δ 172.8, 171.1, 167.7, 165.0, 134.1, 132.9, 132.8, 128.8, 125.6, 125.4, 119.8, 51.5, 43.1, 31.2, 22.6. Tert-butyl((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl) piperidin-4-yl)methyl)carbamate (23)
Obtained using procedure O on 3.44 mmol scale; 1.47 g, 2.8 mmol, yield 81 %, light yellow solid. 1H
NMR (500 MHz, DMSO-d6) δ 11.14 (s, 1H), 11.02 (s, 1H), 8.79 (d, J = 8.5 Hz, 1H), 7.84 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 7.3 Hz, 1H), 6.88 (t, J = 5.7 Hz, 1H), 5.16 (dd, J = 12.9, 5.3 Hz, 1H), 4.10 (q, J = 5.2 Hz, 2H), 3.17 (d, J = 5.2 Hz, 6H), 2.87 – 2.82 (m, 3H), 2.63 – 2.54 (m, 2H), 2.21 – 2.07 (m, 2H), 1.65 – 1.63 (m, 2H), 1.37 (s, 9H).13C NMR (126 MHz, DMSO-d6) δ 172.8, 170.5, 169.9, 168.0, 166.8, 163.2, 159.8, 155.7, 136.4, 131.4, 124.1, 117.9, 115.7, 77.4, 61.8, 53.3, 48.9, 45.5, 35.5, 30.9, 29.6, 28.3, 21.9. HRMS (ESI): m/z calcd for C26H34O7N5 [M+H]+:528.2453; found 528.2448.
2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (24)
obtained using procedure K on 5 mmol scale; 1.2 g, 4.3 mmol, yield 85 %, white solid. The crude product was purified by column chromatography (DCM – MeOH, 0 – 5% MeOH in DCM). 1H NMR (500 MHz, DMSO-d
6) δ 11.16 (s, 1H), 7.96 – 7.92 (m, 1H), 7.79 (d, J = 7.3 Hz, 1H), 7.75 – 7.7.2 (m, 1H), 5.16 (dd, J = 13.0, 5.4 Hz, 1H), 2.91 – 2.85 (m, 1H), 2.63 – 2.58 (m, 1H), 2.53 – 2.51 (m, 1H), 2.07 – 2.04 (m, 1H). 13C NMR (126 MHz, DMSO-d 6) δ 172.8, 169.7, 166.1, 164.0, 156.8 (d, J = 262.3 Hz), 138.0 (d, J = 7.9 Hz), 133.5, 123.0 (d, J = 19.6 Hz), 120.1 (d, J = 3.2 Hz), 117.0 (d, J = 12.6 Hz), 49.1, 30.9, 21.9.
11
Tert-butyl (3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)
carbamate (25)
Obtained using procedure P on 1.03 mmol scale; 266 mg, 0.6 mmol, yield 60 %, yellow solid. 1H NMR (500 MHz, CDCl
3) δ 8.09 (s, 1H), 7.51 – 7.48 (m, 1H), 7.10 (d, J = 7.1 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.31 (s, 1H), 4.91 (dd, J = 12.4, 5.3 Hz, 1H), 4.65 (s, 1H), 3.35 – 3.31 (m, 2H), 3.26 – 3.25 (m, 2H), 2.82 – 2.72 (m, 3H), 2.14 – 2.10 (m, 1H), 1.87 – 1.83 (m, 2H), 1.44 (s, 9H).13C NMR (126 MHz, CDCl 3) δ 171.0, 169.4, 168.3, 167.6, 156.1, 146.7, 136.2, 132.5, 116.5, 111.6, 110.1, 79.5, 48.9, 43.4, 40.0, 31.4, 29.9, 28.4, 22.8. Tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)carbamate (26)
Obtained using procedure P on 1.03 mmol scale; 270 mg, 0.6 mmol, yield 60 %, yellow solid. 1H NMR (500 MHz, CDCl
3) δ 8.37 (s, 1H), 7.47 (dd, J = 8.3, 7.3 Hz, 1H), 7.08 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.23 (d, J = 5.6 Hz, 1H), 4.91 (dd, J = 12.3, 5.4 Hz, 1H), 4.62 (s, 1H), 3.31 – 3.27 (m, 2H), 3.18 – 3.16 (m, 2H), 2.86 – 2.72 (m, 3H), 2.13 – 2.10 (m, 1H), 1.70 – 1.67 (m, 2H), 1.65 – 1.57 (m, 2H), 1.43 (s, 9H).13C NMR (126 MHz, CDCl3) δ 171.2, 169.5, 168.4, 167.6, 156.0, 146.8, 136.1, 132.4, 116.6, 111.5, 109.9, 79.3, 48.8, 42.2, 31.4, 28.4, 27.5, 26.4, 22.8. N1-((4-(5-(3-cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)morpholin-2-yl)methyl)-n5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glutaramide (27)
Obtained using procedure Q1 on 0.27 mmol scale; 75 mg, 0.11 mmol, yield 40 %, yellow solid.1H NMR (500 MHz, CDCl 3) δ 11.10 (s, 1H), 9.40 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 8.46 (s, 1H), 7.77 – 7.75 (m, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.69 – 7.65 (m, 1H), 7.60 – 7.58 (m, 1H), 7.52 (dd, J = 9.9, 7.6 Hz, 2H), 6.07 – 6.05 (m, 1H), 4.97 (dd, J = 12.2, 5.4 Hz, 1H), 3.69 – 3.64 (m, 2H), 3.57 – 3.52 (m, 1H), 3.46 – 3.39 (m, 2H), 3.33 (dd, J = 8.9, 4.9 Hz, 1H), 3.00 – 2.97 (m, 1H), 2.90 – 2.85 (m, 2H), 2.80 – 2.76 (m, 2H), 2.65 – 2.63 (m, 1H), 2.52 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.1 Hz, 2H), 2.15 – 2.13 (m, 1H), 2.07 – 2.03 (m, 2H), 1.87 – 1.85 (m, 2H).13C NMR (126 MHz, CDCl 3) δ 172.1, 171.8, 169.0, 168.8, 166.7, 160.1, 153.3, 150.7, 137.6, 136.5, 136.3, 132.3, 131.6, 131.1, 130.1, 129.5, 125.3, 122.2, 118.7, 118.5, 115.4, 115.0, 112.6, 103.0, 76.8, 74.0, 66.0, 51.6, 49.3, 41.3, 36.6, 35.0, 31.4, 22.7, 21.0, 20.9. HRMS (ESI): m/z calcd for C36H34O7N9 [M+H]+:704.2576; found 704.2571.
4-(5-(3-Cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)-n-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)morpholine-2-carboxamide (28)
Obtained using procedure Q1 on 0.10 mmol scale; 35 mg, 0.05 mmol, yield 50 %, white solid.1H
NMR (500 MHz, CDCl3) δ 10.46 (s, 1H), 9.00 (s, 1H), 8.52 (s, 1H), 7.83 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.60 – 7.54 (m, 2H), 7.51 – 7.48 (m, 1H), 7.10 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.62 (t, J = 5.3 Hz, 1H), 6.41 (t, J = 5.8 Hz, 1H), 4.96 – 4.93 (m, 1H), 4.06 (d, J = 11.5 Hz, 1H), 3.83 (d, J = 10.8 Hz, 2H), 3.70 (d, J = 12.7 Hz, 1H), 3.57 (t, J = 11.4 Hz, 1H), 3.35 (q, J = 6.5 Hz, 2H), 3.31 – 3.28 (m, 2H), 2.97 (t, J = 12.1 Hz, 1H), 2.90 – 2.86 (m, 1H), 2.83 – 2.74 (m, 2H), 2.71 – 2.66 (m, 2H), 2.14 – 2.11 (m, 1H), 1.84 – 1 .79 (m, 2H). 13C NMR (126 MHz, CDCl 3) δ 171.5, 169.3, 169.1, 167.6, 160.0, 153.4, 146.6, 136.4, 136.1, 132.6, 132.4, 131.9, 130.1, 129.6, 127.5, 122.1, 118.8, 116.4, 115.3, 112.6, 111.6, 110.2, 88.8, 74.7, 66.0, 52.0, 48.9, 39.9, 36.2, 31.5, 30.9, 29.2, 22.8. HRMS (ESI): m/z calcd for C34H32O6N9 [M+H]+:662.247; found 662.2467.
4-(5-(3-Cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)-n-((1-(2-((2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)piperidin-4-yl)methyl)morpholine-2-carboxamide (29)
Obtained using procedure Q1 on 0.10 mmol scale; 30 mg, 0.04 mmol, yield 40 %, yellow solid.1H NMR (500 MHz, CDCl 3) δ 11.39 (s, 1H), 10.95 (s, 1H), 8.85 – 8.81 (m, 1H), 8.50 (d, J = 10.7 Hz, 1H), 7.81 (d, J = 6.0 Hz, 1H), 7.73 – 7.68 (m, 2H), 7.59 – 7.53 (m, 3H), 6.48 – 6.47 (m, 1H), 5.04 – 4.99 (m, 1H), 3.99 (d, J = 12.7 Hz, 1H), 3.87 (d, J = 11.0 Hz, 1H), 3.80 – 3.74 (m, 2H), 3.62 – 3.59 (m, 1H), 3.31 – 3.21 (m, 2H), 3.18 – 3.02 (m, 3H), 2.95 – 2.86 (m, 4H), 2.81 – 2.77 (m, 1H), 2.64 – 2.61 (m, 1H), 2.34 – 2.30 (m, 1H), 2.23 – 2.20 (m, 2H), 2.01 – 2.00 (b, 1H), 1.80 – 1.78 (m, 1H), 1.71 – 1.62 (m, 3H), 1.47 – 1.42 (m, 2H).13C NMR (126 MHz, CDCl3) δ 172.0, 170.9, 168.6, 168.2, 166.9, 160.1, 153.4, 150.7, 137.0, 136.1, 132.8, 131.5, 130.1, 129.5, 124.7, 122.3, 119.0, 118.3, 116.0, 115.2, 112.6, 103.3, 74.8, 66.0, 62.1, 53.8, 52.4, 49.2, 48.2, 44.2, 35.2, 31.2, 30.0, 23.1. HRMS (ESI): m/z calcd for C39H39O7N10 [M+H]+:759.2998; found 759.3002.
5-(4-(5-(3-Cyanophenyl)-7h-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-1-yl)-n-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-5-oxopentanamide (30)
Obtained using procedure Q1 on 0.12 mmol scale; 40 mg, 0.06 mmol, yield 50 %, off-white solid.1H NMR (500 MHz, CDCl 3) δ 11.29 (s, 1H), 10.05 (s, 1H), 9.39 (s, 1H), 8.77 (d, J = 8.5 Hz, 1H), 7.80 (s, 1H), 7.75 – 7.68 (m, 2H), 7.59 (d, J = 7.7 Hz, 1H), 7.54 – 7.52 (m, 2H), 7.28 (s, 1H), 4.96 (dd, J = 12.3, 5.3 Hz, 1H), 3.41 – 3.37 (m, 6H), 3.18 (b, 2H), 2.93 – 2.90 (m, 1H), 2.82 – 2.77 (m, 2H), 2.55 (dd, J = 6.8, 5.3 Hz, 2H), 2.42 (t, J = 7.1 Hz, 2H), 2.16 – 2.10 (m, 1H), 2.08 – 2.00 (m, 3H).13C NMR (126 MHz, CDCl 3) δ 171.8, 171.7, 170.4, 169.0, 168.7, 166.7, 160.0, 153.3, 150.5, 137.6, 136.5, 136.4, 132.4, 131.7, 131.1, 130.1, 129.5, 125.3, 122.3, 118.6, 118.5, 115.4, 115.1, 112.7, 49.3, 48.7, 44.7, 40.8, 36.8, 31.9, 31.4, 29.7, 22.7, 20.4. HRMS (ESI): m/z calcd for C35H32O6N9 [M+H]+:674.247; found 674.2466.
N1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-N5-((4-(4-((4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxybenzoyl)morpholin-2-yl) methyl)glutaramide (31)
Obtained using procedure Q1 on 0.11 mmol scale; 32 mg, 0.04 mmol, yield 35 %, white solid.1H
NMR (500 MHz, CDCl3) δ 9.42 (b, 1H), 8.78 (t, J = 7.3 Hz, 1H), 8.43 (dd, J = 12.1, 8.6 Hz, 1H), 8.19 (s, 1H), 7.89 – 7.85 (m, 1H), 7.70 (t, J = 7.9 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H), 6.90 (d, J = 5.7 Hz, 1H), 6.03 (s, 1H), 5.21 (s, 1H), 4.96 – 4.94 (m, 1H), 4.59 – 4.56 (m, 1H), 3.90 (s, 3H), 3.62 – 3.57 (m, 4H), 3.56 – 3.50 (m, 3H), 3.32 – 3.30 (m, 1H), 3.01 – 2.99 (m, 1H), 2.90 (dd, J = 12.9, 2.4 Hz, 1H), 2.76 (d, J = 11.3 Hz, 3H), 2.58 – 2.48 (m, 2H), 2.34 – 2.28 (m, 2H), 2.17 – 2.03 (m, 4H), 1.32 (t, J = 7.2 Hz, 3H).13C NMR (126 MHz, CDCl 3) δ 172.1, 171.6, 170.9, 169.0, 168.0, 166.6, 165.7, 160.2, 158.8, 154.5, 152.2 (d, J = 238.5 Hz), 151.9, 144.3, 137.6, 136.4, 132.01, 131.1, 124.4 (d, J = 270 Hz) 118.6, 115.4, 109.8 (d, J = 4.6 Hz), 105.6, 99.9, 74.6, 66.6, 56.3, 49.3, 44.8, 42.2, 41.1, 36.4, 35.1, 31.3, 22.7, 21.0, 14.4. HRMS (ESI): m/z calcd for C38 H40O9N9F4 [M+H]+:842.288; found 842.2875.
N-((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl) piperidin-4-yl)methyl)-4-((4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxybenzamide (32)
Obtained using procedure Q1 on 0.2 mmol scale; 80 mg, 0.11 mmol, yield 52%, off-wwhite solid.1H NMR
(500 MHz, CDCl3) δ 11.31 (s, 1H), 8.85 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 15.2 Hz, 1H), 8.19 (s, 1H), 7.98 (b, 1H), 7.70 – 7.66 (m, 1H), 7.54 (dd, J = 20.0, 7.2 Hz, 2H), 6.86 (dt, J = 11.7, 5.6 Hz, 1H), 5.27 (b, 1H), 4.94 (dd, J = 11.9, 5.6 Hz, 1H), 3.93 (s, 3H), 3.61 – 3.58 (m, 2H), 3.50 – 3.46 (m, 1H), 3.40 – 3.47 (m, 1H), 3.21 – 3.12 (m, 2H), 2.95 – 2.92 (m, 2H), 2.89 – 2.86 (m, 2H), 2.78 – 2.74 (m, 2H), 2.33 (t, J = 10.1 Hz, 1H), 2.26 (t, J = 10.5 Hz, 1H), 2.18 – 2.15 (m, 1H), 1.78 – 1.76 (m, 2H), 1.65 – 1.63 (m, 3H), 1.30 (t, J = 7.2 Hz, 3H).13C NMR (126 MHz, CDCl 3) δ 171.4, 170.9, 168.3, 168.0, 166.9, 163.5 (d, J = 3.8 Hz), 160.1, 158.7, 155.4 (d, J = 237.5 Hz), 144.0, 137.0, 136.1, 133.1 (d, J = 13.7 Hz), 131.4, 124.9, 124.6 (q, J = 267.9 Hz), 118.3, 116.0, 112.2 (d, J = 13.1 Hz), 111.2 (d, J = 3.3 Hz), 105.5 (d, J = 35.8 Hz), 99.8 (q, J = 32.3 Hz), 62.2, 56.3, 53.9, 49.2, 45.5, 36.4, 35.42, 31.3, 30.0, 29.75, 22.9, 14.4. HRMS (ESI): m/z calcd for C36H38O7N9F4 [M+H]+: 784.2825; found 784.282.
N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)-4-((4-(ethylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-2-fluoro-5-methoxybenzamide (33)
Obtained using procedure Q1 on 0.16 mmol scale; 58 mg, 0.08 mmol, yield 52%, yellow solid.1H NMR (500 MHz, CDCl3) δ 8.69 (s, 1H), 8.45 (d, J = 15.2 Hz, 1H), 8.20 (s, 1H), 7.97 (s, 1H), 7.55 (d, J = 7.1 Hz, 1H), 7.47 – 7.44 (m, 1H), 7.06 (d, J = 7.1 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.25 (t, J = 5.7 Hz, 1H), 5.26 (b, 1H), 4.91 (dd, J = 12.3, 5.3 Hz, 1H), 3.93 (s, 3H), 3.62 – 3.57 (m, 2H), 3.54 – 3.52 (m, 2H), 3.32 (d, J = 5.8 Hz, 2H), 2.88 – 2.80 (m, 1H), 2.77 – 2.69 (m, 2H), 2.13 – 2.09 (m, 1H), 1.76 – 1.74 (m, 4H), 1.32 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl 3) δ 171.3, 169.5, 168.6, 167.6, 163.5 (d, J = 3.8 Hz), 160.2, 158.8, 155.5 (d, J = 237.4 Hz), 146.8, 144.0, 136.1, 133.2 (d, J = 13.9 Hz), 132.5, 124.7 (q, J = 270.4 Hz), 116.7, 112.2 (d, J = 13.1 Hz), 111.5, 111.1 (d, J = 3.5 Hz), 110.0, 105.6 (d, J = 35.3 Hz), 99.9 (q, J = 33.2 Hz), 56.3, 48.9, 42.2, 39.5, 36.4, 31.4, 27.1, 26.7, 22.8, 14.4. HRMS (ESI): m/z calcd for C32H33O6N8F4 [M+H]+:701.2454; found 701.2451.
